Project summary/abstract Type I interferons (IFN) are potent antiviral cytokines whose expression must be tightly regulated to combat infection without driving the development of inflammatory and autoimmune pathologies. Upregulation of type I IFN is pathogenic in a group of monogenic disorders termed type I interferonopathies and the majority are caused by mutations in genes involved in nucleic acid metabolism. Defects in RNA metabolism can result in aberrant activation of innate immune RNA sensors by cellular RNAs, initiating type I interferon expression. Chronic type I IFN signaling activates a signature of interferon stimulated gene (ISG) expression associated with autoimmune and autoinflammatory disease. Furthermore, ISG expression is highly heterogeneous and this heterogeneity directs immune cell state and function. Retinoic acid-inducible gene I (RIG-I) is a RNA innate immune sensor that recognizes double-stranded RNAs with 5-triphosphates ? a unique feature of viral RNA replication ? to initiate type I IFN expression. Mutations in the RNA exosome (35 degradation) cofactor SKIV2L cause the rare multisystem disorder trichohepatoenteric syndrome; and people with this disorder and SKIV2L mutations have a type I IFN signature. In cells that don't express SKIV2L, activation of the unfolded protein response (UPR) also initiates type I IFN expression dependent on a RIG-I mediated signaling. The RNA endoribonuclease Ire1 cleaves ER- localized mRNAs during the UPR as part of Regulated IRE1-Dependent Decay (RIDD), producing RNA fragments with 5-hydroxyl and 2,3-cyclic phosphate ends. We hypothesize that SKIV2L is required during RIDD to target Ire1 cleaved ER-targeted mRNAs for degradation to prevent activation of a RIG-I dependent type I interferon response. Currently, the RNAs that initiate this response and the consequences of the activated ISG program are unknown. The tools needed to comprehensively identify endogenous cleaved RNAs and direct RIG-I-RNA interactions have limited our understanding of endogenous RNA detection by RIG-I. Our aims are to (1) identify Ire1 cleaved RNAs targeted by SKIV2L for degradation to prevent RIG-I-dependent IFN induction using a novel RNA end-sequencing method to directly identify RNAs cleaved by Ire1 in SKIV2L mutant cells during RIDD; and (2) characterize heterogeneous ISG expression induced by direct activation of RIG-I by Ire1 cleaved RNAs using a cross-linking and immunoprecipitation method to capture and sequence RIG-I bound RNAs and single cell RNA sequencing to profile transcriptional and cellular heterogeneity of the ISG response induced by these endogenous RNAs. These studies will facilitate efforts to understand the molecular basis of endogenous RNA detection by RIG-I and contribute to a developing paradigm highlighting the critical role of RNA sensing by innate immune regulators in interferon-mediated autoinflammatory and autoimmune disease.